Tryptophan hydroxylase in rat brainstem extracts is activated 2 to 2.5 fold by ATP and Mg++ in the presence of subsaturating concentrations of the pteridine cofactor 6-methyltetrahydropterins (6MPH4). The activation by ATP-Mg++ is also observed if the natural cofactor tetrahydrobiopterin or the synthetic cofactor dimethyltetrahydropterin is used. The activation requires ATP and Mg++ and is not dependent on cyclic nucleotides. The ATP-Mg++ stimulation is enhanced by calcium and can be blocked by EGTA. REmoval of calmodulin from the hydroxylase containing extracts by affinity chromatography on fluphenazine-Sepharose rendered tryptophan hydroxylase unresponsive to activation by ATP=Mg++. The readdition of calmodulin restored the ATP-Mg++-induced activation in a calcium dependent manner. Drugs which bind to calmodulin also block the ATP-Mg++ effect on tryptophan hydroxylase.